The focus of this project is to understand the pathogenesis of hairy leukoplakia (HL) by characterizing Epstein Barr virus (EBV) gene expression in the lesion, examining the role of individual EBV gene products in modulating epithelial differentiation, and studying regulation of EBV gene expression in this lesion for comparison with that of lymphocytes. We have made significant progress toward achieving each of these aims. The hallmark of HL is high level EBV replication in differentiated epithelium. In the next fiev years,we therefore propose to extend these studies to include characterization of epithelial genes upregulated in HL, and to study the interaction between viral and cellular genes. To date, relatively little is known about the repertoire of EBV gene expression within HL, and almost nothing is known of epithelial gene expression in the lesion. The aims of this proposal are to characterize EBV gene expression in HL; to characterize cellular genes upregulated in Hl, and to characterize the regulation of EBV gene products expressed in HL. In the next five years, using an HlcDNA library we generated, we will continue to expand our knowledge of the repertoire of EBV genes expressed in HL. Using a subtraction cDNA library generated from HL and clinically normal adjacent tissues, we will also characterize cellular genes upregulated in HL. The topography and specificity of expression of these genes will be studied in HL and control tissues by mRNA in situ hybridization. To study the regulation of EBV genes by cellular and viral transactivating proteins, we will induce protein expression by our HL cDNA library. Proteins expressed in the library will be probed with radiolabeled DNA corresponding to promoters of selected EBV genes known to be expressed in HL. Clones encoding the proteins will be isolated from the library, and specificity of the candidate cellular and/or viral DNA binding proteins will be studied using gel retardation assays. The proteins will then be expressed to study their effect on the promoter in luciferase reporter system. Studies of HL offer a unique opportunity to characterize EBV infection of epithelium. Infection of epithelium plays an important role in the EBV live cycle, and understanding the interaction between EBV and cellular genes in the pathogenesis of HL has broader implications for understanding of other EBV-associated lesions as well, including nsaopharyngeal cancer and non-Hodgkin's lymphoma.